A preparation recently developed by the applicants will be used to study the mechanisms controlling the excitation-contraction coupling and the contraction of cardiac cells. Broken single cardiac cells will be obtained by homogenization of adult myocardium of various animal species. Remaining fragments of sarcolemma will be removed by microdissection. Tension will be recorded with a photodiode force transducer from the ends of the skinned (sarcolemma free) cardiac cell (smallest tension detected: one microgram). Sarcomere motion will be recorded with a fast speed movie camera. In the presence of a high total (EGTA) (4.0mM) any Ca2 ion released from the sarcoplasmic reticulum (SR) does not modify the free (Ca2 ion) in the myofilament space. The curve of tension as a function of the free (Ca2 ion) obtained under these conditions represents the direct effect of Ca2 ion on the myofilaments. The effects of free (Mg2 ion), (MgATP), pH and ionic strength on this curve will be studied in order to define the mechanism of contractile activation of the myofilaments. The role of the interaction between cells and of the sarcolemma in the resting tension observed in cardiac muscle will be established by comparing the curve of resting tension as a function of the sarcomere length obtained in skinned cardiac cells to this obtained in the intact muscle. The control of the myoplasmic free (Ca2 ion) with EGTA-Ca buffers will permit the study of the sarcomere length-dependence of the Ca2 ion activation of the myofilaments and of the kinetics of their contractions. The applicants have shown that cyclic contractions are induced by a small variation of free (Ca2 ion) in the presence of a slight buffering with low total (EGTA) (0.050 mM). The demonstration of this Ca2 ion -triggered release of Ca2 ion will be completed by defining the ionic, metabolic and pharmacological factors modulating the amount of Ca2 ion released from the SR. Furthermore the sarcomere length-tension curve for these cyclic contractions due to a Ca2 ion-triggered release of Ca2 ion from the SR will be compared to that obtained for the tonic contraction (by direct activation of teh myofilaments), to investigate the subcellular mechanism of the Starling's law.